PROJECT #2 SUMMARY: Insights into ARDS and VILI pathobiology have been incremental and effective targeted pharmacotherapies have not yet been realized. Project #2 addresses the novel role of NAMPT, the gene encoding nicotinamide phosphoribosyltransferase, in the pathobiology of ARDS and ventilator?induced lung injury (VILI). We identified NAMPT by genomic?intensive approaches utilizing cellular and preclinical models of excessive mechanical stress and VILI. We demonstrated that excessive mechanical stress induces robust NAMPT expression and secretion (extracellular NAMPT or eNAMPT) serves as a novel ARDS biomarker. We have shown that NAMPT exhibits 5' promoter single nucleotide polymorphisms (SNPs) that significantly alter NAMPT promoter activity and confer significantly increased ARDS susceptibility and ARDS severity (reduced ventilator-free days, in- creased ARDS mortality). We determined that eNAMPT is an essential participant in VILI pathobiology directly producing a neutrophilic alveolitis and lung injury whereas reductions in eNAMPT availability (neutralizing anti- bodies, siRNAs, NAMPT+/- mice) dramatically attenuates the severity of lung injury in preclinical VILI /ARDS models. Finally, we demonstrated that NAMPT expression is spatially-localized with robust expression and se- cretion by lung endothelial cells (ECs) with eNAMPT a novel ligand for the Toll?like receptor 4 (TLR4) inducing NF?B transcriptional activities and inflammatory lung injury. Although eNAMPT is clearly an attractive ARDS/VILI target, critical gaps remain in the understanding of NAMPT-mediated lung pathobiology, issues which need to be addressed for robust translation to an ICU therapy. Project #2 will address these key gaps focusing on mechanical stress-challenged lung EC (a major source of secreted eNAMPT), on eNAMPT contri- bution to increases in vascular permeability (a major therapeutic target in ARDS), and on the critical influence of eNAMPT binding to lung EC TLR4 in VILI development. Project #2 Specific Aims (SAs) are designed to ad- dress these gaps with SA #1 elucidating mechanical stress-mediated genetic and epigenetic regulation of NAMPT expression (transcription factors, CpG demethylation, 3'UTR miRNA binding, NAMPT SNPs). Based on exciting preliminary data, SA #2 will define regulation of eNAMPT secretion by caspase-mediated cleavage and ABC transporters. With Core B (Molecular Biology & Genetics Core) and Core D (Protein Chemistry Core), SA #3 will define structure/function mechanisms involved in NAMPT binding of TLR4 and the influence of TLR4 and NAMPT coding SNPs on ligand?receptor interactions. Finally, utilizing preclinical VILI/ARDS models, in- cluding a novel conditional EC?specific and lung epithelium-specific NAMPT KO mice (Core C: Pre-clinical An- imal Model Core), SA #4 will translate SA #1- #3 data into actionable information to attenuate VILI/ARDS and define the impact of reduced NAMPT expression and secretion (STAT5/HIF2? inhibitors), eNAMPT elimination (neutralizing antibodies), and TLR4 antagonism (peptide inhibitors). Project #2 will advance understanding of NAMPT participation in VILI/ARDS and promote the application of individualized therapies for the critically ill.